Dual Control of Host Actin Polymerization by a Legionella Effector Pair

Figure 4

The LegK2/VipA suppression pair does not meet the definition of metaeffector. (a) Cellular localization of ARPC1B/LegK2 and EEA1/VipA proteins in HeLa cells transfected by pDEST27-legK2 or peGFP-N-vipA. GST-LegK2 proteins were detected by immunofluorescence with anti-GST antibodies (Sigma, green), and ARPC1B was detected by anti-ARPC1B antibodies (SantaCruz; red). VipA-GFP proteins were detected thanks to GFP fusion and EEA1 by anti-EEA1 antibodies (Cell Signaling Technology). Scale bar represents 10 μm. (b) Calnexin-GFP expressing D. discoideum cells were infected at with L. pneumophila WT Paris transformed with pMMB207c-Ptac-HA-legK2 or pMMB207c-Ptac-HA-vipA plasmid. Infected cells were fixed 1 hour postinfection, and HA-tagged proteins were labelled by immunofluorescence with HA-antibodies. Legionella DNA was stained with DAPI. Scale bar represents 6 μm. (c) GFP-trap copurification assay of GFP-tagged VipA with GST-tagged LegK2 proteins. HeLa cells were cotransfected by pDEST27 or pDEST27-legK2 and peGFP-N-vipA or peGFP-C-vipA. GFP or GFP-tagged VipA were purified on GFP-Trap agarose beads, and finally, total lysates (L) and eluted fractions (E) were immunoblotted with both anti-GST and anti-GFP antibodies. (d) In vitro phosphorylation assays of 6His-VipA by LegK2 detected by Western blot with antiphosphothreonine antibodies. The 6His-VipA fusion protein purified from E. coli BL21(DE3) was incubated with purified GST-LegK2 in the presence of 100 μM ATP. The myelin basic protein (MBP), known to be a substrate of LegK2 [31], was used as a positive control of phosphorylation. Proteins were then separated by SDS-PAGE and detected with antiphosphothreonine antibodies.

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